CN111700865A - Live vaccine, live vaccine freeze-dried powder, live vaccine protective agent, and preparation method and application thereof - Google Patents
Live vaccine, live vaccine freeze-dried powder, live vaccine protective agent, and preparation method and application thereof Download PDFInfo
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- CN111700865A CN111700865A CN202010788239.5A CN202010788239A CN111700865A CN 111700865 A CN111700865 A CN 111700865A CN 202010788239 A CN202010788239 A CN 202010788239A CN 111700865 A CN111700865 A CN 111700865A
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- live vaccine
- protective agent
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- 239000003223 protective agent Substances 0.000 title claims abstract description 92
- 238000002360 preparation method Methods 0.000 title claims abstract description 37
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- 239000001963 growth medium Substances 0.000 claims abstract description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 43
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- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 42
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- 229960005055 sodium ascorbate Drugs 0.000 claims abstract description 29
- 235000010378 sodium ascorbate Nutrition 0.000 claims abstract description 29
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims abstract description 29
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims abstract description 29
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- 229920003081 Povidone K 30 Polymers 0.000 description 6
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 5
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0275—Salmonella
-
- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Biochemistry (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of live vaccine protective agents, and particularly relates to a live vaccine, live vaccine freeze-dried powder, a live vaccine protective agent, and a preparation method and application thereof. The invention provides a live vaccine protective agent, which comprises water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone, sodium ascorbate, sodium L-glutamate, 199 culture medium and water for injection. The live vaccine protective agent provided by the invention can increase the survival rate and the preservation period of bacteria and improve the protection function of the live vaccine protective agent on the bacteria under the high-temperature condition; and the conditions of animal allergy and immune failure caused by improper storage of the live vaccine can be avoided.
Description
Technical Field
The invention belongs to the technical field of live vaccine protective agents, and particularly relates to a live vaccine, live vaccine freeze-dried powder, a live vaccine protective agent, and a preparation method and application thereof.
Background
The heat sensitivity of active organisms such as bacteria and related products thereof is always a technical bottleneck for long-term storage of live vaccines. China has wide breadth, large climate difference, uneven economic level, and the phenomenon of short-term exposure to high-temperature environment (even more than 40 ℃) in the process of vaccine transportation and use still exists, so that higher requirements are put forward on the heat resistance of the live vaccine.
At present, in the preparation process of the vaccine, the traditional freeze-drying protective agents in the bacterial live vaccine are mainly sucrose and gelatin protective agents, the formula is simple, the freeze-drying survival rate is low, the protection function is poor (especially at a higher temperature), and the storage life is short. The vaccine prepared by the protective agent has strict preservation conditions and is not heat-resistant in the using process. If the preservation method is improper, the bacterial survival rate of the vaccine is reduced, and even most of thalli are dead or the endotoxin generated by disintegration is one of the factors generating anaphylactic reaction; in addition, the storage and transportation and the use at remote places are difficult due to low-temperature storage. Because the vaccine is not stored properly, the animal is allergic and killed, so that the immunity is failed, and the effect of protecting the animal cannot be achieved.
Disclosure of Invention
The invention aims to provide a live vaccine, live vaccine freeze-dried powder, a live vaccine protective agent, a preparation method and an application thereof, wherein the live vaccine protective agent can increase the survival rate and the storage life of bacteria and improve the protection function of the live vaccine protective agent on the bacteria under the high-temperature condition; and the conditions of animal allergy and immune failure caused by improper storage of the live vaccine can be avoided.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a live vaccine protective agent, which comprises water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone, sodium ascorbate, sodium L-glutamate, 199 culture medium and water for injection; each 100mL of the live vaccine protective agent comprises 1.5-9 g of water-soluble gelatin, 5-30 g of oligosaccharide, 1-24 g of sorbitol, 0.1-6 g of polyvinylpyrrolidone, 0.5-12 g of sodium ascorbate, 0.5-18 g of L-sodium glutamate and 0.5-3 g of 199 culture medium.
Preferably, the oligosaccharide is sucrose.
The invention provides a preparation method of the live vaccine protective agent, which comprises the following steps:
mixing water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone and the first residual water for injection, and sterilizing at high temperature to obtain a first raw material;
mixing and filtering the L-sodium glutamate, the sodium ascorbate, the 199 culture medium and the second residual water for injection to obtain a second raw material;
mixing the first raw material and the second raw material, and supplementing the rest with third water for injection to obtain the live vaccine protective agent.
Preferably, the high-temperature sterilization temperature is 116-118 ℃, and the time is 15-25 min.
The invention provides an application of the live vaccine protective agent in the technical scheme or the live vaccine protective agent prepared by the preparation method in the technical scheme in improving the heat resistance of the yak paratyphoid live vaccine.
The invention provides a live vaccine, which comprises a strain and the live vaccine protective agent in the technical scheme or the live vaccine protective agent prepared by the preparation method in the technical scheme; the strain is salmonella dublin STM8002-550 strain (Zhangxiaming, Pi Zheng, Li Hui Ju, Li Guangxi, Cheng Yan Shu. cultivation of the yak paratyphoid attenuated strain and research on immunity thereof, Chinese veterinary science and technology, 1991). In the present invention, the Salmonella dublin STM8002-550 strain is deposited and provided by the veterinary institute of livestock in Gansu province.
The invention provides a preparation method of the live vaccine, which comprises the following steps:
inoculating seed liquid of salmonella dublin STM8002-550 strain to a culture medium for culture to obtain zymogen liquid;
centrifuging the zymocyte liquid to obtain bacterial sludge;
mixing the bacterial sludge with normal saline to obtain bacterial liquid for preparing the seedlings;
and (3) uniformly mixing the live vaccine protective agent and the bacterial liquid for vaccine preparation according to a proportion to obtain the live vaccine.
Preferably, the culture medium comprises a nutrient broth medium; the inoculation amount of the inoculation is 1-3% of the volume of the culture medium; the volume ratio of the live vaccine protective agent to the bacterial liquid for vaccine preparation is 1-1: 1 to 5.
The invention also provides a live vaccine freeze-dried powder, and the preparation method of the live vaccine freeze-dried powder comprises the step of drying the live vaccine of the technical scheme to obtain the live vaccine freeze-dried powder.
Preferably, the drying comprises vacuum freeze drying; the vacuum freeze-drying procedure is as follows: the vacuum freeze-drying comprises a first freeze-drying, a second freeze-drying and a third freeze-drying; the temperature of the first freeze drying is below minus 40 ℃, the vacuum degree is 0mpa, and the time is 1.5-3.5 h; the temperature of the second freeze drying is-15 to-4 ℃, the vacuum degree is 10 to 25mpa, and the time is 12 to 18 hours; the temperature of the third freeze drying is 20-28 ℃, the vacuum degree is 10-25 mpa, and the time is 7-10 h.
The invention provides a live vaccine protective agent, which comprises water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone, sodium ascorbate, sodium L-glutamate, 199 culture medium and water for injection; each 100mL of the live vaccine protective agent comprises 1.5-9 g of water-soluble gelatin, 5-30 g of oligosaccharide, 1-24 g of sorbitol, 0.1-6 g of polyvinylpyrrolidone, 0.5-12 g of sodium ascorbate, 0.5-18 g of L-sodium glutamate and 0.5-3 g of 199 culture medium. The components and the content of the live vaccine protective agent provided by the invention are reasonably matched, so that the live vaccine is heat-resistant and storage-resistant. The polyvinylpyrrolidone k30 in the heat-resistant substance can prevent the active component from being denatured in the freezing and drying processes, and plays a role in supporting and filling; the oligosaccharide can permeate into cells, so that the osmotic pressure increased by freezing is reduced, the cell membrane of bacteria can be protected, the bacteria are in a dormant state, the life activities of the bacteria during drying and storage period are reduced, the heat resistance of the bacteria is greatly enhanced, the survival rate of the microorganisms can be improved, uniform suspension is formed, the moisture relieving effect is achieved, and the low-temperature protection function and the dehydration protection effect can be achieved; the water-soluble gelatin is a high molecular compound, has a protection effect on microorganisms, can promote the microorganisms to sublimate to form a heat-resistant framework, and blocks heat conduction and heat radiation; sodium L-glutamate, a thermolabile substance, is a basic constituent unit of protein and is also the best filler; the sodium ascorbate blocks oxidation chain reaction in the freeze-dried sample, inhibits the activity of oxidase and prevents the sample from oxidative deterioration in the freeze drying and storage processes; sorbitol can improve the sublimation rate of water molecules in the freeze-drying process and improve the stability of freeze-dried products; 199 the small molecular amino acid in the culture medium can slow down the change of the pH value of the solution and protect the active components from being damaged; 199 in the freeze drying and storage process of potassium dihydrogen phosphate and disodium hydrogen phosphate in the culture medium, adjusting the pH value of the biological product to the most stable region of the active substance; under the combined action of the components, the live vaccine protective agent provided by the invention can increase the survival rate and the preservation period of bacteria, and improves the protection function of the live vaccine protective agent on the bacteria under the high-temperature condition; and the conditions of animal allergy and immune failure caused by improper storage of the live vaccine can be avoided. Test results show that the live vaccine protective agent provided by the invention has good heat-resistant protection effect.
Detailed Description
The invention provides a live vaccine protective agent, which comprises water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone, sodium ascorbate, sodium L-glutamate, 199 culture medium and water for injection; each 100mL of the live vaccine protective agent comprises 1.5-9 g of water-soluble gelatin, 5-30 g of oligosaccharide, 1-24 g of sorbitol, 0.1-6 g of polyvinylpyrrolidone, 0.5-12 g of sodium ascorbate, 0.5-18 g of L-sodium glutamate and 0.5-3 g of 199 culture medium.
The sources of the above raw materials are not particularly limited in the present invention, and conventional commercial products well known to those skilled in the art may be used, for example, polyvinylpyrrolidone, sorbitol, mannitol, sodium L-glutamate, sodium ascorbate, sucrose, water-soluble gelatin, trehalose are preferably available from Solarbio corporation; 199 the medium is preferably purchased from GIBCO.
The invention provides a live vaccine protective agent, which comprises a heat-resistant substance, a thermolabile substance and water for injection.
The raw material of the heat-resistant substance in the live vaccine protective agent comprises 1.5-9 g of water-soluble gelatin, preferably 3-9 g, and more preferably 9g based on 100mL of the live vaccine protective agent. In the invention, the water-soluble gelatin is a high molecular compound which has a protection effect on microorganisms, can promote the sublimation of the high molecular compound to form a heat-resistant framework and block heat conduction and heat radiation.
Based on the mass of the water-soluble gelatin, the raw material of the heat-resistant substance in the live vaccine protective agent provided by the invention comprises 5-30 g of oligosaccharide, preferably 10-30 g, and more preferably 30 g. In the present invention, the oligosaccharide is preferably sucrose. In the invention, the oligosaccharide-sucrose can permeate into cells, reduce osmotic pressure increased by freezing, protect bacterial cell membranes, enable bacteria to be in a dormant state, reduce life activities of bacteria drying and storage period, further greatly enhance heat resistance of the bacteria, improve survival rate of microorganisms, form uniform suspension and play a role in relieving moisture, wherein the sucrose can play a role in low-temperature protection and dehydration protection.
Based on the mass of the water-soluble gelatin, the raw material of the heat-resistant substance in the live vaccine protective agent provided by the invention comprises 1-24 g of sorbitol, preferably 8-24 g, and more preferably 24 g. In the invention, the sorbitol can improve the sublimation rate of water molecules in the freeze-drying process and improve the stability of a freeze-dried product.
Based on the mass of the water-soluble gelatin, the raw material of the heat-resistant substance in the live vaccine protective agent provided by the invention comprises 0.1-6 g of polyvinylpyrrolidone, preferably 0.2-0.6 g, and more preferably 0.6 g; the polyvinylpyrrolidone is preferably polyvinylpyrrolidone k30(PVP-k 30). In the invention, polyvinylpyrrolidone (PVP) in the heat-resistant protective agent can prevent the active component from being denatured in the freezing and drying processes, and plays a role in supporting and filling.
Based on the mass of the water-soluble gelatin, the raw material of the thermolabile substance in the live vaccine protective agent provided by the invention comprises 0.5-12 g of sodium ascorbate, preferably 4-12 g, and more preferably 12 g. In the invention, the sodium ascorbate blocks oxidation chain reaction in the freeze-dried sample, inhibits oxidase activity and prevents the sample from oxidative deterioration in the freeze-drying and storage processes.
The raw material of the thermolabile substance in the live vaccine protective agent provided by the invention comprises 0.5-18 g L-sodium glutamate, preferably 6-18 g, and more preferably 18g by taking the mass of the water-soluble gelatin as a reference. In the present invention, sodium L-glutamate is a basic constituent unit of protein, and is also a preferable filler.
Based on the mass volume concentration of the water-soluble gelatin, the raw material of the thermolabile substance in the live vaccine protective agent provided by the invention comprises 0.5-3 g of 199 culture medium, preferably 1-3 g, and more preferably 3 g. In the invention, the buffer salt in the 199 culture medium can buffer the change of the pH value of the solution, and the micromolecule amino acid protects the active components from being damaged; 199 the pH of the biological product is adjusted to the region of most stable active substance during the freeze-drying and storage process of the potassium dihydrogen phosphate and the disodium hydrogen phosphate in the culture medium.
The rest is made up by water for injection.
The invention provides a preparation method of the live vaccine protective agent, which comprises the following steps:
mixing water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone and the first residual water for injection, and sterilizing at high temperature to obtain a first raw material;
mixing and filtering the L-sodium glutamate, the sodium ascorbate, the 199 culture medium and the second residual water for injection to obtain a second raw material;
mixing the first raw material and the second raw material, and supplementing the rest with third water for injection to obtain the live vaccine protective agent.
According to the invention, water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone k30 and the first residual water for injection are mixed and sterilized at high temperature to obtain the first raw material. In the present invention, the amount of the first remaining water for injection is preferably 30 ml; the high-temperature sterilization temperature is preferably 112-118 ℃, more preferably 114-118 ℃, and most preferably 116-118 ℃; the time for high-temperature sterilization is preferably 15min to 30min, more preferably 20min to 30min, and most preferably 25 min. The high-temperature sterilization temperature specified in the present invention can avoid the deterioration of the sugar in the above-mentioned materials due to an excessively high temperature.
The invention mixes and filters L-sodium glutamate, sodium ascorbate, 199 culture medium and the second residual water for injection to obtain the second raw material. In the present invention, the amount of the remaining water for injection is preferably 30 ml; the filtration is preferably membrane filtration; the pore size of the filter membrane for the filter membrane filtration is preferably 0.22 μm. In the invention, the substance components are easy to damage after high-temperature sterilization, and the problem of substance component damage is avoided by selecting a filtration sterilization mode.
The invention mixes the first raw material and the second raw material, and the shortage is complemented by the third water for injection to obtain the live vaccine protective agent. In the present invention, the mixing method is not limited at all, and can be a method known to those skilled in the art. In the present invention, the first material and the second material are mixed so that the components in the solution are uniform.
The invention provides an application of the live vaccine protective agent in the technical scheme or the live vaccine protective agent prepared by the preparation method in the technical scheme in improving the heat resistance of the yak paratyphoid live vaccine.
The invention provides a live vaccine, which comprises a strain and the live vaccine protective agent in the technical scheme or the live vaccine protective agent prepared by the preparation method in the technical scheme; the strain is salmonella dublin STM8002-550 strain (Zhangxiaming, Pi Zheng, Li Hui Ju, Li Guangxi, Cheng Yan Shu. cultivation of the yak paratyphoid attenuated strain and research on immunity thereof, Chinese veterinary science and technology, 1991). In the present invention, the combination of the strain and the live vaccine protective agent can provide the vaccine with the advantages of heat resistance and storage resistance.
The invention provides a preparation method of the live vaccine, which comprises the following steps:
inoculating seed liquid of salmonella dublin STM8002-550 strain to a culture medium for culture to obtain zymogen liquid;
centrifuging the zymocyte liquid to obtain bacterial sludge;
mixing the bacterial sludge with normal saline to obtain bacterial liquid for preparing the seedlings;
and (3) uniformly mixing the live vaccine protective agent and the bacterial liquid for vaccine preparation according to a proportion to obtain the live vaccine.
The method comprises the step of inoculating seed liquid of salmonella dublin STM8002-550 strain to a culture medium for culture to obtain zymogen liquid. In the present invention, the culture medium preferably comprises a nutrient broth medium; the nutrient broth culture medium is preferably prepared according to the standard in the veterinary biological product code of the people's republic of China; the inoculation amount of the inoculation is preferably 1-3% of the volume of the culture medium, more preferably 1-2%, and most preferably 1%; the pH value of the culture is 7.2-7.4, more preferably 7.2-7.3, and most preferably 7.2. In the present invention, the carbon source supplement solution in the culture process is preferably 40% glucose. In the invention, 40 percent of glucose is added in the culture process to supplement a carbon source required by bacteria, and the glucose is utilized by the bacteria to generate CO2Dissolving in water to form carbonic acid, thereby adjusting the pH value of the solution and playing a role in reducing the pH value of the solution. In the present invention, the mode of the culture preferably includes a fermentation culture or an aeration culture; the aeration culture preferably comprises two stages of standing culture and aeration culture; the fermentation culture refers to the inoculation of salmonella berlin STM8002-550 strain liquid culture medium and shaking culture at 185r/min at a specific temperature. Or the aeration culture refers to inoculating salmonella berlin STM8002-550 strain liquid culture medium, performing static culture at a specific temperature, and then introducing a certain amount of filtered air for culture, wherein the culture preferably comprises static culture and aeration culture; the temperature of the fermentation culture is preferably 36-38 ℃, more preferably 36.5-37.5 ℃, and most preferably 37 ℃; the time of fermentation culture is preferably 16-20 h, more preferably 17-20 h, and most preferably 18-20 h; the aeration rate of the aeration culture is preferably 0.5kg/cm2～3.5kg/cm2More preferably 0.5kg/cm2～2.0kg/cm2Most preferably 0.5kg/cm2～1.0kg/cm2(ii) a The time of the aeration culture is preferably 16 to 20 hours, more preferably 16.5 to 20 hours, and most preferably 17 to 20 hours; the temperature of the stationary culture is preferably temperaturePreferably 38 ℃ to 39.5 ℃, more preferably 38.5 ℃ to 39 ℃, and most preferably 38.5 ℃; the time for the static culture is preferably 3 to 5 hours, more preferably 3.5 to 4 hours, and most preferably 4 hours. In the present invention, it is preferable that a sterilized antifoaming agent is added to the culture solution before the aeration culture; the defoaming agent is preferably vegetable oil such as castor oil and soybean oil; the amount of the defoaming agent added is preferably 0.1% to 0.22%, more preferably 0.16% to 0.2%, and most preferably 0.2%. In the invention, the defoaming agent is added during aeration culture, so that the foam generated by gas introduced into the culture solution can be eliminated, and the loss of the culture solution is reduced.
After the zymophyte liquid is obtained, the zymophyte liquid is centrifuged to obtain bacterial sludge. In the invention, the rotation speed of the centrifugation is preferably 3000 r/min-12000 r/min, more preferably 3300 r/min-12000 r/min, and most preferably 3500 r/min-12000 r/min; the time for centrifugation is preferably 10min to 30min, more preferably 15min to 30min, and most preferably 20min to 30 min. In the invention, when a tubular high-speed continuous centrifuge is adopted, the rotating speed is 12000r/min (the machine type only has 12000r/min rotating speed).
After the bacterial sludge is obtained, the bacterial sludge is mixed with normal saline to obtain the bacterial liquid for preparing the seedlings. In the present invention, the physiological saline is preferably sterilized physiological saline; the dosage ratio of the physiological saline to the bacterial sludge is preferably 30: 1-150: 1, more preferably 50: 1-120: 1, and most preferably 80: 1-100: 1. In the present invention, the mixing method is not limited at all, and may be a method known to those skilled in the art.
After obtaining the bacterial liquid for vaccine preparation, the invention uniformly mixes the live vaccine protective agent and the bacterial liquid for vaccine preparation according to the proportion to obtain the live vaccine. In the invention, the volume ratio of the live vaccine protective agent to the bacterial liquid for vaccine preparation is 1-1: 1 to 5, more preferably 1 to 1: 2 to 5, most preferably 1: 5. In the present invention, the mixing method is not limited at all, and may be a method known to those skilled in the art.
The invention also provides a live vaccine freeze-dried powder, and the preparation method of the live vaccine freeze-dried powder comprises the step of drying the live vaccine of the technical scheme to obtain the live vaccine freeze-dried powder.
The live vaccine prepared by the technical scheme is dried to obtain the live vaccine freeze-dried powder. The drying preferably comprises vacuum freeze drying; the vacuum drying procedure is as follows: the vacuum freeze-drying preferably includes a first freeze-drying, a second freeze-drying and a third freeze-drying.
In the present invention, the temperature of the first freeze-drying is preferably-50 to-35 ℃, more preferably 45 to-35 ℃, and most preferably-43 to-40 ℃; the degree of vacuum of the first drying is preferably 0 mpa; the second freeze drying time is preferably 1.5-4 h, more preferably 2-4 h, and most preferably 2-3 h; the present invention preferably reduces to the first freeze-drying temperature at a rate of 1 c per minute.
In the present invention, the temperature of the second freeze-drying is preferably-15 to-4 ℃, more preferably-12 to-3 ℃, and most preferably-10 to-8 ℃; the vacuum degree of the second freeze drying is preferably 10-25 mpa, more preferably 10-20 mpa, and most preferably 10-15 mpa; the second freeze drying time is preferably 12-18 h, more preferably 12-17 h, and most preferably 12-14 h; after the first freeze-drying is completed, the present invention preferably increases the temperature from the first freeze-drying temperature to the second freeze-drying temperature at a rate of 2 ℃ per hour.
In the invention, the temperature of the third freeze drying is preferably 20-28 ℃, more preferably 22-27 ℃, and most preferably 25-26 ℃; the vacuum degree of the third drying is preferably 10-25 mpa, more preferably 10-20 mpa, and most preferably 10-15 mpa; the time is preferably 7-10 h, more preferably 7-9 h, and most preferably 7.5-8.5 h; the present invention preferably increases the temperature from the second freeze-drying temperature to the third freeze-drying temperature at a rate of 10 ℃ per 2 hours. In the embodiment of the present invention, the vacuum freeze-drying procedure is preferably:
putting the vaccine bottle containing the bacterial liquid into a box body of a freeze dryer with the temperature of 0-4 ℃, reducing the temperature to below-40 ℃ at the speed of 1 ℃ per minute, and maintaining the temperature below-40 ℃ for 2 hours; starting vacuumizing until the vacuum degree is 10mpa, gradually increasing the temperature of the product from below-40 ℃ to-10 ℃, and maintaining for 12-14 h; heating to 10 ℃ every 2h, and keeping the temperature of the product at 25 ℃ for about 8 h; the whole freeze-drying process takes 28-30 h. The heat-resistant live vaccine freeze-dried powder for the paratyphoid fever of the yak, which is prepared by adopting the heat-resistant protective agent, avoids the formation of ice crystals, reduces the physical damage of bacterial cell membranes, reduces the loss rate in the freeze-drying process, reduces the death rate of thalli and anaphylactic reaction generated by metabolic byproducts, and ensures that the live vaccine freeze-dried powder can be stored for 2 months at 25 ℃ and stored for 24 months at 2-8 ℃.
In order to further illustrate the present invention, a live vaccine, a lyophilized powder of a heat-resistant live vaccine, a heat-resistant protective agent for a live vaccine, and a preparation method and application thereof provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparing raw materials: 1.5g of water-soluble gelatin, 5g of cane sugar, 5g of sorbitol, 0.1g of polyvinylpyrrolidone k30, 5g L-sodium glutamate, 1.5g of sodium ascorbate, 0.5g of 199 culture medium, and the balance of distilled water to 100 mL.
Mixing 1.5g of water-soluble gelatin, 5g of cane sugar, 5g of sorbitol, 0.1g of polyvinylpyrrolidone k30 and 30mL of water for injection, and sterilizing at 25 ℃ for 25min to obtain a first raw material;
mixing 5g L-sodium glutamate, 1.5g sodium ascorbate, 0.5g 199 culture medium and 30mL water for injection, and filtering to obtain a second raw material;
mixing the first material and the second material, and supplementing the rest with sterilized water for injection to obtain live vaccine protective agent.
Example 2
Preparation of bacterial liquid for preparing vaccine of paratyphoid live vaccine of yak
The seed liquid was prepared according to the "regulations for veterinary biological products for the people's republic of china" (good quality year edition, Ministry of agriculture of the people's republic of china-Beijing: chemical industry Press, 2000, hereinafter referred to as "regulations").
Inoculating the seed solution 2% into sterilized 5 bottles filled with 200mL nutrient broth culture medium, fermenting and culturing at 37 deg.C and 185r/min for 17h, centrifuging the bacterial solution at 3500r/min for 20min, collecting all bacterial sludge, suspending with sterilized normal saline, and mixing well to obtain bacterial solution for preparing seedling.
Example 3
Preparing raw materials: 1.5g of water-soluble gelatin, 5g of cane sugar, 0.1g of PVP-K30, 0.5g of 199 culture medium, 1.5g of sodium ascorbate, 3g L-sodium glutamate, 4g of sorbitol and the balance of distilled water to 100 mL. The preparation method is the same as that of example 1.
Example 4
Preparing raw materials: 1.5g of water-soluble gelatin, 5g of sucrose, 0.1g of PVP-K30, 0.5g of 199 culture medium, 2g of sodium ascorbate, 5g L-sodium glutamate, 4g of sorbitol and the balance of distilled water to 100 mL. The preparation method is the same as that of example 1.
Example 5
Preparing raw materials: 1.5g of water-soluble gelatin, 5g of cane sugar, 0.1g of PVP-K30, 0.5g of 199 culture medium, 2g of sodium ascorbate, 3g L-sodium glutamate, 4g of sorbitol and the balance of distilled water to 100 mL. The preparation method is the same as that of example 1.
Example 6
Preparing raw materials: 6g of water-soluble gelatin, 20g of cane sugar, 0.4g of PVP-K30, 2g of 199 culture medium, 8g of sodium ascorbate, 12g L-sodium glutamate, 16g of sorbitol and the balance of distilled water to 100L. The preparation method is the same as that of example 1.
Example 7
Preparing raw materials: 9g of water-soluble gelatin, 30g of cane sugar, 0.6g of PVP-K30, 3g of 199 culture medium, 12g of sodium ascorbate, 18g L-sodium glutamate, 24g of sorbitol and the balance of distilled water to 100 mL. The preparation method is the same as that of example 1.
Example 8
Preparing live vaccine freeze-dried powder:
(1) preparing a bacterium liquid for bacterium preparation: seed liquid of Salmonella dublin STM8002-550 strain is prepared according to the requirements of the regulations.
(2) Inoculating seed liquid of Salmonella dublin STM8002-550 strain into common broth according to 3% of total amount of culture medium, and fermenting and culturing at 37 deg.C for 17h at 185 r/min; and adding 10-100 ml of sterilized 40% sterilized glucose to supplement a carbon source according to the condition of pH rise in the culture process, and controlling the pH value to 7.2-7.4. And (3) after the culture is finished, immediately centrifuging, removing supernatant, collecting bacterial sludge, adding a proper amount of sterilized normal saline, and uniformly stirring to obtain the bacterial liquid for preparing the seedlings.
(3) Respectively mixing the live vaccine protective agent prepared in the embodiment 3-5 with the yak paratyphoid Dublin salmonella liquid 1: 1 proportion to obtain live vaccines 3, 4 and 5.
(4) Respectively mixing the protective agent solution prepared in the embodiment 6-7 with the yak paratyphoid Dublin salmonella liquid 1: 3. 1:5 to obtain live vaccines 6 and 7.
(5) And (3) carrying out freeze drying on the live vaccine by using a vacuum freeze dryer under the same freeze-drying curve to obtain live vaccine freeze-dried powder 3, 4, 5, 6 and 7.
The freeze drying method comprises the following steps: putting live vaccine into a box at 0-4 ℃, reducing the temperature to below-40 ℃ at the speed of 1 ℃ per minute, and maintaining the temperature below-40 ℃ for 2 hours; (ii) a Starting to vacuumize, then gradually increasing the temperature of the product from below-40 ℃ to-10 ℃, and maintaining for 12-14 h; heating to 10 deg.C every 2h, maintaining the temperature of the product at 25 deg.C for about 8h, and taking out. The whole freeze-drying process takes 28 to 30 hours.
Comparative example 1
Weighing 6g of water-soluble gelatin, diluting to 100mL with water for injection to obtain 6% water-soluble gelatin stock solution, adjusting pH to 7.2 with 2M sodium hydroxide solution, and sterilizing at 116 deg.C for 30min to obtain live vaccine protectant.
Comparative example 2
Weighing PVP-K3012g, diluting to 100mL with water for injection to obtain 12% PVP-K30 liquid, adjusting pH to 7.2 with 2M sodium hydroxide solution, and sterilizing at 116 deg.C for 30min to obtain live vaccine protectant.
Comparative example 3
Weighing 10g of mannitol, dissolving with water for injection, diluting to 100mL, and filtering and sterilizing with a 0.22 mu m filter membrane to obtain the live vaccine protective agent.
Comparative example 4
Weighing 10g of sorbitol, dissolving with water for injection, diluting to 100mL, and filtering and sterilizing with a 0.22-micron filter membrane to obtain the live vaccine protective agent.
Comparative example 5
Weighing 15g of sucrose, dissolving with water for injection, diluting to 100mL, and filtering and sterilizing with a 0.22-micron filter membrane to obtain the live vaccine protective agent.
Comparative example 6
Weighing 15g of trehalose, dissolving the trehalose in water for injection, diluting to 100mL, and filtering and sterilizing through a 0.22 mu m filter membrane to obtain the live vaccine protective agent.
Comparative example 7
Weighing 15g of L-sodium glutamate, dissolving with water for injection, diluting to 100mL, and filtering and sterilizing with 0.22 μm filter membrane to obtain the live vaccine protective agent.
Comparative example 8
Weighing 15g of sodium ascorbate, dissolving with water for injection, diluting to 100mL, and filtering with 0.22 μm filter membrane for sterilization to obtain the live vaccine protective agent.
Comparative example 9
Weighing 199 culture medium 2g, dissolving with water for injection, diluting to 100mL, and filtering with 0.22 μm filter membrane for sterilization to obtain live vaccine protective agent.
In example 1 and comparative examples 1 to 9, PVP-k30 (polyvinylpyrrolidone k30), sorbitol, mannitol, sodium L-glutamate, sodium ascorbate, sucrose, water-soluble gelatin and trehalose were produced by Solambio corporation; 199 the medium was produced by GIBCO.
Application example 1
Freeze thaw test
After 1mL of the bacterial liquid prepared in example 2 and the live vaccine protective agents prepared in example 1 and comparative examples 1 to 9 were mixed in equal amounts (the final concentration of the protective agent is shown in Table 1), the mixture was placed at 2 to 8 ℃ for 20min, and then stored at-40 ℃ for 4 h. Meanwhile, bacteria liquid without protective agent matrix is set as a reference. After each frozen bacterium solution is thawed, viable bacteria are counted together with a control sample before freezing and thawing according to the national animal pharmacopoeia (the national animal pharmacopoeia of China: 2010 edition, three parts/China animal pharmacopoeia committee-Beijing: China agricultural publishing Co., Ltd., 2011.3, hereinafter referred to as the Chinese animal pharmacopoeia), and the frozen bacterium solution storage rate is calculated. The results are shown in Table 1.
TABLE 1 results of viable count and viability in freeze thaw tests
From the results in table 1, 9 matrices were known as protecting agents, among which: the bacterial storage rate of the L-sodium glutamate and the sucrose is the highest and can reach more than 60 percent at most; trehalose, mannitol, water-soluble gelatin, 199 culture medium and sodium ascorbate have the next highest bacteria survival rate, and the highest bacteria survival rate is more than 50%. The live vaccine protective agent provided by the invention can obviously improve viable count and freeze-thaw bacteria survival rate after thawing.
Application example 2
Freeze drying test
10mL of the bacterial liquid for vaccine preparation in the example 2 is mixed with the live vaccine protective agent prepared in the example 1 in equal amount; respectively taking 10mL of the bacterial liquid for preparing the vaccine in the example 2, equivalently mixing the bacterial liquid with a basic protective agent (water-soluble gelatin with the mass volume concentration of 1.5% and sucrose with the mass volume concentration of 5%) and the live vaccine protective agents prepared in the comparative examples 1-9 (the final concentration of the protective agent is shown in the table 2), quantitatively subpackaging the mixture by 1mL, and freeze-drying according to the appendix of Chinese veterinary pharmacopoeia. Meanwhile, 1.5% of water-soluble gelatin and 5% of sucrose are used as a reference. Respectively taking samples before and after freeze-drying, and counting viable bacteria and calculating the survival rate of freeze-dried bacteria according to Chinese veterinary pharmacopoeia. The results are shown in Table 2.
The method of vacuum freeze drying comprises the following steps: putting live vaccine into a box at 0-4 ℃, reducing the temperature to below-40 ℃ at the speed of 1 ℃ per minute, and maintaining the temperature below-40 ℃ for 2 hours; starting to vacuumize, then gradually increasing the temperature of the product from below-40 ℃ to-10 ℃, and maintaining for 12-14 h; the temperature is raised by 10 ℃ every 2h, and the temperature is maintained at 25 ℃ for about 8 h. The whole freeze-drying process takes 28 to 30 hours.
TABLE 2 results of viable count and viability of samples before and after lyophilization
The results in table 2 show that 0.5% 199 culture medium, 3% sodium L-glutamate and 1.5% sodium ascorbate are added into 1.5% water-soluble gelatin and 5% sucrose protective agent respectively, so that the freeze-drying bacterial storage rate can be obviously improved; 1.5 percent of water-soluble gelatin, 5 percent of cane sugar and 4 percent of sorbitol can ensure that the freeze-dried bacteria storage rate reaches 67.5 percent. Therefore, the addition of 0.1% PVP, 0.5% 199 culture medium, 3% sodium L-glutamate and 1.5% sodium ascorbate to 1.5% water-soluble gelatin and 5% sucrose protectant also has the effect of improving the bacteria survival rate. The live vaccine protective agent provided by the invention can obviously improve the number of live bacteria and the survival rate of freeze-dried bacteria after freeze-drying.
Application example 3
The live vaccine lyophilized powders 3, 4, 5, 6, and 7 prepared in example 8 and a blank control (a conventional protective agent, namely, a bacterial solution for preparing vaccine, a water-soluble gelatin sucrose solution (a live vaccine protective agent containing 12% by mass of water-soluble gelatin and 40% by mass of sucrose) were mixed uniformly in a volume ratio of 1:7 to serve as a control for lyophilized live vaccine) were subjected to a comparative test of heat resistance according to a method specified by the viable count in the ordinary agar plate culture in the pharmacopoeia of beasts, chinese (2010 edition), and the results are shown in tables 3 and 4.
TABLE 3 results of physical Properties and residual Water determination of lyophilized live vaccine and control vaccine
TABLE 4 viable count results for lyophilized live vaccines in different storage environments and storage times
As can be seen from the above results, the lyophilized live vaccine produced according to example 8 has no significant difference in physical properties and moisture content after lyophilization, compared to the conventional lyophilized vaccine; but shows more stable heat-resistant performance than the conventional freeze-dried vaccine in the storage life test of the vaccine. Meanwhile, the heat-resistant live vaccine for paratyphoid cattle produced by the protective agent in example 7 has more remarkable heat-resistant effect, the number of live bacteria is reduced by only 15 percent when the vaccine is placed at 37 ℃ for 7 days, 2 percent when the vaccine is stored at 25 ℃ for 60 days, and 1 percent when the vaccine is stored at 4 ℃ for 24 months. Therefore, the vaccine 7 prepared by the formula of the heat-resistant protective agent in the example 7 in the example 8 in the application of the vaccine has better heat-resistant protective effect.
As is clear from the above-mentioned examples, the live vaccine protective agent provided by the present invention has a good heat-resistant protective effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A live vaccine protective agent is characterized by comprising water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone, sodium ascorbate, sodium L-glutamate, 199 culture medium and water for injection; each 100mL of the live vaccine protective agent comprises 1.5-9 g of water-soluble gelatin, 5-30 g of oligosaccharide, 1-24 g of sorbitol, 0.1-6 g of polyvinylpyrrolidone, 0.5-12 g of sodium ascorbate, 0.5-18 g of L-sodium glutamate and 0.5-3 g of 199 culture medium.
2. A live vaccine protection agent according to claim 1, wherein the oligosaccharide is sucrose.
3. A method for preparing a live vaccine protective agent according to claim 1 or 2, comprising the steps of:
mixing water-soluble gelatin, oligosaccharide, sorbitol, polyvinylpyrrolidone and the first residual water for injection, and sterilizing at high temperature to obtain a first raw material;
mixing and filtering the L-sodium glutamate, the sodium ascorbate, the 199 culture medium and the second residual water for injection to obtain a second raw material;
mixing the first raw material and the second raw material, and supplementing the rest with third water for injection to obtain the live vaccine protective agent.
4. The preparation method according to claim 3, wherein the high-temperature sterilization is carried out at a temperature of 116 ℃ to 118 ℃ for 15min to 25 min.
5. The use of the live vaccine protective agent of claim 1 or 2 or the live vaccine protective agent obtained by the preparation method of claim 3 or 4 for improving the heat resistance of the live vaccine of paratyphus yak.
6. A live vaccine, characterized in that the live vaccine comprises a strain and the live vaccine protective agent of claim 1 or 2 or the live vaccine protective agent prepared by the preparation method of claim 3 or 4; the strain is Salmonella dublin STM8002-550 strain.
7. A method for preparing a live vaccine according to claim 6, comprising the steps of:
inoculating seed liquid of salmonella dublin STM8002-550 strain to a culture medium for culture to obtain zymogen liquid;
centrifuging the zymocyte liquid to obtain bacterial sludge;
mixing the bacterial sludge with normal saline to obtain bacterial liquid for preparing the seedlings;
and (3) uniformly mixing the live vaccine protective agent and the bacterial liquid for vaccine preparation according to a proportion to obtain the live vaccine.
8. The method of claim 7, wherein the culture medium comprises a nutrient broth medium; the inoculation amount of the inoculation is 1-3% of the volume of the culture medium; the volume ratio of the live vaccine protective agent to the bacterial liquid for vaccine preparation is 1-1: 1 to 5.
9. A lyophilized live vaccine powder, which is characterized in that the preparation method of the lyophilized live vaccine powder comprises the step of drying the live vaccine of claim 6 to obtain the lyophilized live vaccine powder.
10. A live vaccine lyophilized powder according to claim 9, wherein the drying comprises vacuum freeze drying; the vacuum freeze-drying procedure is as follows: the vacuum freeze-drying comprises a first freeze-drying, a second freeze-drying and a third freeze-drying; the temperature of the first freeze drying is below minus 40 ℃, the vacuum degree is 0mpa, and the time is 1.5-3.5 h; the temperature of the second freeze drying is-15 to-4 ℃, the vacuum degree is 10 to 25mpa, and the time is 12 to 18 hours; the temperature of the third freeze drying is 20-28 ℃, the vacuum degree is 10-25 mpa, and the time is 7-10 h.
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| CN202010788239.5A CN111700865A (en) | 2020-08-07 | 2020-08-07 | Live vaccine, live vaccine freeze-dried powder, live vaccine protective agent, and preparation method and application thereof |
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| CN202010788239.5A CN111700865A (en) | 2020-08-07 | 2020-08-07 | Live vaccine, live vaccine freeze-dried powder, live vaccine protective agent, and preparation method and application thereof |
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| US4147772A (en) * | 1976-02-03 | 1979-04-03 | Merck & Co., Inc. | Vaccine stabilizer |
| CN1927395A (en) * | 2006-08-25 | 2007-03-14 | 长春百克生物科技有限公司 | Method for preparation of freeze-dried chickenpox vaccine |
| CN101912608A (en) * | 2010-08-17 | 2010-12-15 | 北京海淀中海动物保健科技公司 | Method for producing paratyphus living vaccine for piglets |
| CN102793927A (en) * | 2012-08-29 | 2012-11-28 | 郑州后羿制药有限公司 | Swine fever vaccine heat-resistant freeze-drying protective agent, and preparation method and application thereof |
| CN106215186A (en) * | 2016-08-02 | 2016-12-14 | 甘肃省畜牧兽医研究所 | Sore mouth virus live-vaccine heat-proof protective agent and lyophilized powder thereof and the preparation method of this lyophilized powder |
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2020
- 2020-08-07 CN CN202010788239.5A patent/CN111700865A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4147772A (en) * | 1976-02-03 | 1979-04-03 | Merck & Co., Inc. | Vaccine stabilizer |
| CN1927395A (en) * | 2006-08-25 | 2007-03-14 | 长春百克生物科技有限公司 | Method for preparation of freeze-dried chickenpox vaccine |
| CN101912608A (en) * | 2010-08-17 | 2010-12-15 | 北京海淀中海动物保健科技公司 | Method for producing paratyphus living vaccine for piglets |
| CN102793927A (en) * | 2012-08-29 | 2012-11-28 | 郑州后羿制药有限公司 | Swine fever vaccine heat-resistant freeze-drying protective agent, and preparation method and application thereof |
| CN106215186A (en) * | 2016-08-02 | 2016-12-14 | 甘肃省畜牧兽医研究所 | Sore mouth virus live-vaccine heat-proof protective agent and lyophilized powder thereof and the preparation method of this lyophilized powder |
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